Material and methods
Specimens were collected from various parts of Norway in 2019, either
from native stands or from cultivated plants of known origin (Figure 1,
Table1). Inflorescences were collected and dried for studies of pollen
morphology, and herbarium specimens were sampled and prepared for
documentation, deposited in herbarium BG.
DNA ploidy levels were measured for the studied specimens except samples
of A. rupicola , H. lancifolia , H. neglecta ,H. subpinnata , and S. aucuparia (Table 2). The ploidy
levels were measured using flow cytometry analysed by Plant Cytometry
Services (Schijndel, The Netherlands) following the procedure given in
Andersen et al. (2016).
Anthers were treated with 10% KOH to remove cell content, followed by
acetolysis to remove cellulose (Fægri et al., 1989). The pollen was
stained using fuchsine and mounted in glycerol. Using light microscope
(LM) the polar axis (P) and the equatorial axis (E) of 100 pollen grains
of each specimen were measured. The P/E ratio was calculated following
Boyd and Dickson (1987). In total 1600 pollen from 16 different
specimens were measured. To assess and compare the shape, central
tendency, variability of the samples, and to look for outliers in 16
different specimens, the side-by-side boxplots were made of the lengths
of the polar axis (P), equatorial axis (E), and the calculated ratio
(P/E) of the 100 measured pollen for each specimen (Figure 2).
Scanning Electron Microscope (SEM) was used to investigate the surface
structure of the pollen and to aid the description of pollen of the
specimens. Pollen was applied with a brush, carefully sprinkled over
carbon tape, and glued to an aluminium rivet. Following by coating with
a thin layer of gold and palladium to make the sample electrically
conductive throughout. Individual pollen grains were visualised with a
Zeiss Supra 55VP scanning electron microscope with 5.00 kV, 30 µm
aperture with a working distance to the lens ~4 mm. From
the resulting pictures, morphological characteristics of the pollen
surface were recorded (Table 3).
Principal Component Analysis (PCA) was first run with the three measures
pollen characters, the polar axis (P), equatorial (E) axis, and their
ratio (P/E). A second analysis with the nine different characters of
pollen morphology; including the measured characters and the
morphological characters of the pollen surface as given in Table 3. The
analyses were performed with centring and standardization of characters
and visualized in CANOCO 5 (Ter Braak and Šmilauer, 2012).